Methods to increase nucleotide signals by raman scattering

ABSTRACT

The methods and apparatus disclosed herein concern nucleic acid sequencing by enhanced Raman spectroscopy. In certain embodiments of the invention, nucleotides are covalently attached to Raman labels before incorporation into a nucleic acid. In other embodiments, unlabeled nucleic acids are used. Exonuclease treatment of the nucleic acid results in the release of labeled or unlabeled nucleotides that are detected by Raman spectroscopy. In alternative embodiments of the invention, nucleotides released from a nucleic acid by exonuclease treatment are covalently cross-linked to nanoparticles and detected by surface enhanced Raman spectroscopy (SERS), surface enhanced resonance Raman spectroscopy (SERRS) and/or coherent anti-Stokes Raman spectroscopy (CARS). Other embodiments of the invention concern apparatus for nucleic acid sequencing.

RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 10/099,287, filed on Mar. 14, 2002; and acontinuation-in-part of U.S. patent application Ser. No. 09/962,555,filed Sep. 24, 2001.

FIELD OF THE INVENTION

The present methods and apparatus relate to the fields of molecularbiology and genomics. More particularly, the methods and apparatusconcern nucleic acid sequencing.

BACKGROUND

Genetic information is stored in the form of very long molecules ofdeoxyribonucleic acid (DNA), organized into chromosomes. The humangenome contains approximately three billion bases of DNA sequence. ThisDNA sequence information determines multiple characteristics of eachindividual. Many common diseases are based at least in part onvariations in DNA sequence.

Determination of the entire sequence of the human genome has provided afoundation for identifying the genetic basis of such diseases. However,a great deal of work remains to be done to identify the geneticvariations associated with each disease. That would require DNAsequencing of portions of chromosomes in individuals or familiesexhibiting each such disease, in order to identify specific changes inDNA sequence that promote the disease. Ribonucleic acid (RNA), anintermediary molecule in processing genetic information, may also besequenced to identify the genetic bases of various diseases.

Existing methods for nucleic acid sequencing, based on detection offluorescently labeled nucleic acids that have been separated by size,are limited by the length of the nucleic acid that can be sequenced.Typically, only 500 to 1,000 bases of nucleic acid sequence can bedetermined at one time. This is much shorter than the length of thefunctional unit of DNA, referred to as a gene, which can be tens or evenhundreds of thousands of bases in length. Using current methods,determination of a complete gene sequence requires that many copies ofthe gene be produced, cut into overlapping fragments and sequenced,after which the overlapping DNA sequences may be assembled into thecomplete gene. This process is laborious, expensive, inefficient andtime-consuming. It also typically requires the use of fluorescent orradioactive labels, which can potentially pose safety and waste disposalproblems.

More recently, methods for nucleic acid sequencing have been developedinvolving hybridization to short oligonucleotides of defined sequenced,attached to specific locations on DNA chips. Such methods may be used toinfer short nucleic acid sequences or to detect the presence of aspecific nucleic acid in a sample, but are not suited for identifyinglong nucleic acid sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the disclosedembodiments of the invention. The embodiments of the invention may bebetter understood by reference to one or more of these drawings incombination with the detailed description of specific embodiments of theinvention presented herein.

FIG. 1 illustrates an exemplary apparatus 10 (not to scale) and methodfor nucleic acid 13 sequencing, using nucleotides 16 covalently attachedto Raman labels.

FIG. 2 illustrates an exemplary apparatus 100 (not to scale) and methodfor nucleic acid 13 sequencing in which the released nucleotides 130 arecovalently attached to nanoparticles 140 prior to detection by surfaceenhanced Raman spectroscopy (SERS) 180.

FIG. 3 illustrates another exemplary apparatus 210 (not to scale) fornucleic acid 13 sequencing.

FIG. 4 shows the Raman spectra of all four deoxynucleosidemonophosphates (dNTPs) at 100 mM concentration, using a 100 milliseconddata collection time. Characteristic Raman emission peaks for as shownfor each different type of nucleotide. The data were collected withoutsurface-enhancement or labeling of the nucleotides.

FIG. 5 shows SERS detection of 1 nM guanine, obtained from dGMP by acidtreatment according to Nucleic Acid Chemistry, Part 1, L. B. Townsendand R. S. Tipson (Eds.), Wiley-Interscience, New York, 1978.

FIG. 6 shows SERS detection of 10 nM cytosine, obtained from dCMP byacid treatment.

FIG. 7 shows SERS detection of 100 nM thymine, obtained from dTMP byacid treatment.

FIG. 8 shows SERS detection of 100 pM adenine, obtained from dAMP byacid treatment.

FIG. 9 shows a comparative SERS spectrum of a 500 nM solution ofdeoxyadenosine triphosphate covalently labeled with fluorescein (uppertrace) and unlabeled dATP (lower trace). The dATP-fluorescein wasobtained from Roche Applied Science (Indianapolis, Ind.). A strongincrease in the SERS signal was detected in the fluorescein labeleddATP.

FIG. 10 shows the SERS detection of a 0.9 nM (nanomolar) solution ofadenine. The detection volume was 100 to 150 femtoliters, containing anestimated 60 molecules of adenine.

FIG. 11 shows the SERS detection of a rolling circle amplificationproduct, using a single-stranded, circular M13 DNA template.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The disclosed methods and apparatus are of use for the rapid, automatedsequencing of nucleic acids. In particular embodiments of the invention,the methods and apparatus are suitable for obtaining the sequences ofvery long nucleic acid molecules of greater than 1,000, greater than2,000, greater than 5,000, greater than 10,000 greater than 20,000,greater than 50,000, greater than 100,000 or even more bases in length.Advantages over prior art methods include the ability to read longnucleic acid sequences in a single sequencing run, greater speed ofobtaining sequence data, decreased cost of sequencing and greaterefficiency in operator time required per unit of sequence data.

In various embodiments of the invention, sequence information may beobtained during the course of a single sequencing run, using a singlenucleic acid molecule. In other embodiments of the invention, multiplecopies of a nucleic acid molecule may be sequenced in parallel orsequentially to confirm the nucleic acid sequence or to obtain completesequence data. In alternative embodiments of the invention, both thenucleic acid molecule and its complementary strand may be sequenced toconfirm the accuracy of the sequence information. In variousembodiments, a nucleic acid to be sequenced may be attached, eithercovalently or non-covalently to a surface. In particular embodiments,nucleotides may be released from a surface-attached nucleic acid, forexample by exonuclease treatment. Released nucleotides may betransported, for example, through a microfluidic system to a Ramandetector, to allow detection of released nucleotides without backgroundRaman signals from the nucleic acid, exonuclease and/or other componentsof the system.

In certain embodiments of the invention, the nucleic acid to besequenced is DNA, although it is contemplated that other nucleic acidscomprising RNA or synthetic nucleotide analogs could be sequenced aswell. The following detailed description contains numerous specificdetails in order to provide a more thorough understanding of thedisclosed embodiments of the invention. However, it will be apparent tothose skilled in the art that the embodiments of the invention may bepracticed without these specific details. In other instances, devices,methods, procedures, and individual components that are well known inthe art have not been described in detail herein.

In various embodiments of the invention, unlabeled nucleotides may bedetected by Raman spectroscopy, for example by surface enhanced Ramanspectroscopy (SERS), surface enhanced resonance Raman spectroscopy(SERBS), coherent anti-Stokes Raman spectroscopy (CARS) or other knownRaman detection techniques. In alternative embodiments, nucleotides maybe covalently attached to Raman labels to enhance the Raman signal. Insome embodiments, labeled nucleotides may be incorporated into a newlysynthesized nucleic acid strand using standard nucleic acidpolymerization techniques. Typically, either a primer of specificsequence or one or more random primers is allowed to hybridize to atemplate nucleic acid. Upon addition of a polymerase and labelednucleotides, the Raman labeled nucleotides are covalently attached tothe 3′ end of the primer, resulting in the formation of a labelednucleic acid strand complementary in sequence to the template. Thelabeled strand may be separated from the unlabeled template, for exampleby heating to about 95° C. or other known methods. The two strands maybe separated from each other by techniques well known in the art. Forexample, the primer oligonucleotide may be covalently modified with abiotin residue and the resulting biotinylated nucleic acid may beseparated by binding to an avidin or streptavidin coated surface.

In alternative embodiments of the invention, either labeled or unlabeledsingle-stranded nucleic acid molecules may be digested with one or moreexonucleases. The skilled artisan will realize that the disclosedmethods are not limited to exonucleases per se, but may utilize anyenzyme or other reagent capable of sequentially removing nucleotidesfrom at least one end of a nucleic acid. In certain embodiments of theinvention, labeled or unlabeled nucleotides are sequentially releasedfrom the 3′ end of the nucleic acid. After separation from the nucleicacid, the nucleotides are detected by a Raman detection unit.Information on sequentially detected nucleotides is used to compile asequence of the nucleic acid. Nucleotides released from the 3′ end of anucleic acid may be transported down a microfluidic flow path past aRaman detector. In particular embodiments, the detector is capable ofdetecting labeled or unlabeled nucleotides at the single molecule level.The order of detection of the nucleotides by the Raman detector is thesame as the order in which the nucleotides are released from the 3′ endof the nucleic acid. The sequence of the nucleic acid can thus bedetermined by the order in which released nucleotides are detected.Where a complementary strand is sequence, the template strand will becomplementary in sequence according to standard Watson-Crick hydrogenbond base-pairing (i.e., A-T and G-C or A-U and G-C, depending onwhether DNA or RNA is sequenced).

In certain alternative embodiments, a tag molecule may be added to areaction chamber or flow path upstream of the detection unit. The tagmolecule binds to and tags free nucleotides as they are released fromthe nucleic acid molecule. This post-release tagging avoids problemsthat are encountered when the nucleotides of the nucleic acid moleculeare tagged before their release into solution. For example, the use ofbulky Raman label molecules may provide steric hindrance when eachnucleotide incorporated into a nucleic acid molecule is labeled beforeexonuclease treatment, reducing the efficiency and increasing the timerequired for the sequencing reaction.

In certain embodiments of the invention, each of the four types ofnucleotide may be attached to a distinguishable Raman label. In otherembodiments of the invention, only the purine nucleotides (cytosineand/or thymine and/or uracil) may be labeled. In one exemplaryembodiment, the labeled nucleotides may comprise biotin-labeleddeoxycytidine-5′-triphosphate (biotin-dCTP) and digoxigenin-labeleddeoxyuridine-5′-triphosphate (digoxigenin-dUTP). In alternativeembodiments, no nucleotides are labeled and the unlabeled nucleotidesare identified by Raman spectroscopy.

In specific embodiments of the invention, the Raman signals may beenhanced by covalent attachment of nucleotides to nanoparticles.Nanoparticles may be prepared as discussed below and activated byattachment of highly reactive groups, for example epoxide groups, usingknown methods. For example, nanoparticles may be coated with3-glycidoxypropyltrimethoxysilane (GOP). GOP contains a terminal highlyreactive epoxide group. The use of highly reactive groups such asepoxides allows for rapid formation of covalent bonds betweennanoparticles and nucleotides. In some embodiments, the nucleotides maybe released from a nucleic acid by exonuclease activity and then reactedwith nanoparticles to allow covalent bond formation. Various methods maybe employed to allow sufficient time for covalent bonds to form beforethe nanoparticle-nucleotide complex. For example, a solution containingreleased nucleotides and activated nanoparticles may be transported bymicrofluidic flow down a relatively long flow path, allowing sufficienttime for covalent bond formation to occur before thenucleotide-nanoparticle complex passes in front of the Raman detector.Flow rate may be further decreased by use of a fluid of high viscosity,for example a glycerol solution. Methods of microfluidic flow of highviscosity solutions are known in the art.

Alternatively, a cyclic process may be employed, wherein a nanoparticleis first allowed to bind to the 3′ end of a nucleic acid. Thenanoparticle and attached nucleotide may be released from the 3′ end ofthe nucleic acid by exonuclease activity or chemical treatment. Forexample, the phosphodiester bond attaching the terminal nucleotide tothe nucleic acid may be cleaved by treatment with acid or base. Theelectron-withdrawing effect of the attached nanoparticle may render theterminal phosphodiester bond particularly labile to cleavage, allowingremoval of a single nucleotide at a time. Following release, anothernanoparticle may be reacted with the 3′ end of the nucleic acid and theprocess repeated in a cycle. Alternatively, a 3′ exonuclease may be usedto release the nucleotide-nanoparticle complex. Steric hindrance fromthe nanoparticle with exonuclease activity may be avoided by using alinker arm to attach the reactive group (e.g., epoxide) to thenanoparticle. Attachment of the nanoparticle before release of theterminal nucleotide would allow ample time for covalent bond formation.In other alternative embodiments of the invention, the rate ofexonuclease activity may be adjusted to coordinate the rates of releaseof nucleotides and their covalent attachment to nanoparticles. Forexample, a reaction chamber containing a nucleic acid and exonucleasemay be temperature controlled to a reduced temperature, of between 0° C.and room temperature. Once an appropriate temperature has beendetermined, the nucleotides released by exonuclease activity may enter aflow path where they are mixed with reactive nanoparticles at anelevated temperature, between room temperature and 100° C. The elevatedtemperature would increase the rate of reactivity of nucleotide andnanoparticle. In various embodiments, temperature ranges from about 0°C. to 5° C., 5° C. to 10° C., 10° C. to 15° C., 15° C. to 20° C. or 20°C. to 25° C. may be used to regulate exonuclease activity. In certainembodiments, temperature ranges from about 20° C. to 25° C., 25° C. to30° C., 30° C. to 35° C., 35° C. to 40° C., 40° C. to 45° C., 45° C. to50° C., 50° C. to 55° C., 55° C. to 60° C., 60° C. to 65° C., 65° C. to70° C., 70° C. to 75° C., 75° C. to 80° C. or 80° C. to 95° C. may beused to regulate the rate of covalent bond formation betweennanoparticle and nucleotide. It is well within the routine skill in theart to assay reaction rates as a function of temperature and to selectappropriate temperature ranges to coordinate the rates of exonucleaseactivity and nucleotide-nanoparticle cross-linking.

In some embodiments of the invention, the nanoparticles are silver orgold, but other types of nanoparticles known to provide surface enhancedRaman signals are contemplated. The nanoparticles may either be singlenanoparticles, aggregates of nanoparticles, or some mixture of singleand aggregated nanoparticles. In certain embodiments, a linker compoundmay be used to attach the nucleotides to the nanoparticles. The linkercompound may be between 1 to 100 nanometers (nm), 2 to 90 nm, 3 to 80nm, 4 to 70 nm, 5 to 60 nm, 10 to 50 nm, 15 to 40 nm or 20 to 30 nm inlength. In certain embodiments, the linker compound may be between 1 to50, 1 to 5, 2 to 10, 10 to 20 nm or about 5 nm in length. In otherembodiments, two or more nanoparticles may be attached together usinglinker compounds.

The nanoparticle-nucleotide complexes may pass through a flow-throughcell where they are detected by SERS, SERRS and/or CARS using a Ramandetection unit. In some alternative embodiments of the invention, thenucleotides may be unmodified, while in other alternative embodimentsthe nucleotides may be modified with one or more Raman labels. Incertain embodiments, each type of nucleotide may be attached to adistinguishable Raman label. In other embodiments only pyrimidines maybe labeled.

DEFINITIONS

As used herein, “a” or “an” may mean one or more than one of an item.

As used herein, “operably coupled” means that there is a functionalinteraction between two or more units. For example, a detector may be“operably coupled” to a flow-through cell if the detector is arranged sothat it may detect analytes, such as nucleotides, as they pass throughthe flow-through cell.

“Nucleic acid” encompasses DNA, RNA, single-stranded, double-stranded ortriple stranded and any chemical modifications thereof. Virtually anymodification of the nucleic acid is contemplated. As used herein, asingle stranded nucleic acid may be denoted by the prefix “ss”, a doublestranded nucleic acid by the prefix “ds”, and a triple stranded nucleicacid by the prefix “ts.” A “nucleic acid” may be of almost any length,from 10, 20, 30, 40, 50, 60, 75, 100, 150, 200, 250, 300, 400, 500, 600,700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000,6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, 30,000, 40,000, 50,000,75,000, 100,000, 150,000, 200,000, 500,000, 1,000,000, 1,500,000,2,000,000, 5,000,000 or even more bases in length, up to a full-lengthchromosomal DNA molecule.

A “nucleoside” is a molecule comprising a purine or pyrimidine base(adenine—“A”, cytosine—“C”, guanine—“G”, thymine—“T” or uracil—“U”) orany chemical modification or structural analog thereof, covalentlyattached to a pentose sugar such as deoxyribose, ribose or derivativesor analogs of pentose sugars.

A “nucleotide” refers to a nucleoside further comprising at least onephosphate group covalently attached to the pentose sugar. In someembodiments of the invention, the nucleotides are ribonucleosidemonophosphates or deoxyribonucleoside monophosphates, although it isanticipated that nucleoside diphosphates or triphosphates could beproduced and detected. In other embodiments of the invention,nucleosides may be released from the nucleic acid molecule. It iscontemplated that various substitutions or modifications may be made inthe structure of the nucleotides, so long as they are capable of beingincorporated into a nucleic acid by polymerase activity and released byan exonuclease or equivalent reagent. In embodiments of the inventioninvolving one or more labels attached to one or more types ofnucleotide, the label may be attached to any portion of the nucleotide,such as the base, the sugar or the phosphate groups or their analogs.The terms “nucleotide” and “labeled nucleotide” encompass, but are notlimited to, all non-naturally nucleotide complexes, such asnucleotide-nanoparticle complexes and nucleotide-label complexes.

A “Raman label” may be any organic or inorganic molecule, atom, complexor structure capable of producing a detectable Raman signal, includingbut not limited to synthetic molecules, dyes, naturally occurringpigments such as phycoerythrin, organic nanostructures such as C₆₀,buckyballs and carbon nanotubes, metal nanostructures such as gold orsilver nanoparticles or nanoprisms and nano-scale semiconductors such asquantum dots. Numerous examples of Raman labels are disclosed below. Theskilled artisan will realize that such examples are not limiting, andthat “Raman label” encompasses any organic or inorganic atom, molecule,compound or structure known in the art that can be detected by Ramanspectroscopy.

Nucleic Acids

Nucleic acid molecules to be sequenced may be prepared by any techniqueknown in the art. In certain embodiments of the invention, the nucleicacids are naturally occurring DNA or RNA molecules. Virtually anynaturally occurring nucleic acid may be prepared and sequenced by thedisclosed methods including, without limit, chromosomal, mitochondrialand chloroplast DNA and ribosomal, transfer, heterogeneous nuclear andmessenger RNA (mRNA). Methods for preparing and isolating various formsof nucleic acids are known. (See, e.g., Guide to Molecular CloningTechniques, eds. Berger and Kimmel, Academic Press, New York, N.Y.,1987; Molecular Cloning; A Laboratory Manual, 2nd Ed., eds. Sambrook,Fritsch and Maniatis, Cold Spring Harbor Press, Cold Spring Harbor,N.Y., 1989). The methods disclosed in the cited references are exemplaryonly and any variation known in the art may be used. In cases wheresingle stranded DNA (ssDNA) is to be sequenced, an ssDNA may be preparedfrom double stranded DNA (dsDNA) by any known method. Such methods mayinvolve heating dsDNA and allowing the strands to separate, or mayalternatively involve preparation of ssDNA from dsDNA by knownamplification or replication methods, such as cloning into M13. Any suchknown method may be used to prepare ssDNA or ssRNA. As discussed above,one of the two strands of double-stranded DNA may be separated, forexample, by biotin labeling and attachment to avidin or streptavidinusing known techniques.

Although certain embodiments of the invention concern preparation ofnaturally occurring nucleic acids, virtually any type of nucleic acidthat can serve as a substrate for an exonuclease or equivalent reagentcould potentially be sequenced. For example, nucleic acids prepared byvarious amplification techniques, such as polymerase chain reaction(PCR™) amplification, could be sequenced. (See U.S. Pat. Nos. 4,683,195,4,683,202 and 4,800,159.) Nucleic acids to be sequenced mayalternatively be cloned in standard vectors, such as plasmids, cosmids,BACs (bacterial artificial chromosomes) or YACs (yeast artificialchromosomes). (See, e.g., Berger and Kimmel, 1987; Sambrook et al.,1989.) Nucleic acid inserts may be isolated from vector DNA, forexample, by excision with appropriate restriction endonucleases,followed by agarose gel electrophoresis. Methods for isolation of insertnucleic acids are well known.

Isolation of Single Nucleic Acid Molecules

In certain embodiments of the invention, the nucleic acid molecule to besequenced is a single molecule of ssDNA or ssRNA. A variety of methodsfor selection and manipulation of single nucleic acid molecules may beused, for example, hydrodynamic focusing, micro-manipulator coupling,optical trapping, or a combination of these and similar methods. (See,e.g., Goodwin et al., 1996, Acc. Chem. Res. 29:607-619; U.S. Pat. Nos.4,962,037; 5,405,747; 5,776,674; 6,136,543; 6,225,068.)

In certain embodiments of the invention, microfluidics or nanofluidicsmay be used to sort and isolate nucleic acid molecules. Hydrodynamicsmay be used to manipulate the movement of nucleic acids into amicrochannel, microcapillary, or a micropore. In one embodiment of theinvention, hydrodynamic forces may be used to move nucleic acidmolecules across a comb structure to separate single nucleic acidmolecules. Once the nucleic acid molecules have been separated,hydrodynamic focusing may be used to position the molecules within areaction chamber. A thermal or electric potential, pressure or vacuumcan also be used to provide a motive force for manipulation of nucleicacids. In exemplary embodiments of the invention, manipulation ofnucleic acids for sequencing may involve the use of a channel blockdesign incorporating microfabricated channels and an integrated gelmaterial (see U.S. Pat. Nos. 5,867,266 and 6,214,246).

In another embodiment of the invention, a sample containing the nucleicacid molecule may be diluted prior to coupling to an immobilizationsurface. In exemplary embodiments of the invention, the immobilizationsurface may be in the form of magnetic or non-magnetic beads or otherdiscrete structural units. At an appropriate dilution, each bead willhave a statistical probability of binding zero or one nucleic acidmolecule. Beads with one attached nucleic acid molecule may beidentified using, for example, fluorescent dyes and flow cytometersorting or magnetic sorting. Depending on the relative sizes anduniformity of the beads and the nucleic acids, it may be possible to usea magnetic filter and mass separation to separate beads containing asingle bound nucleic acid molecule. In other embodiments of theinvention, multiple nucleic acids attached to a single bead or otherimmobilization surface may be sequenced.

In alternative embodiments of the invention, a coated fiber tip may beused to generate single molecule nucleic acids for sequencing (e.g.,U.S. Pat. No. 6,225,068). In other alternative embodiments, theimmobilization surfaces may be prepared to contain a single molecule ofavidin or other cross-linking agent. Such a surface could attach asingle biotinylated nucleic acid molecule to be sequenced. Thisembodiment is not limited to the avidin-biotin binding system, but maybe adapted to any known coupling system.

In other alternative embodiments of the invention, an optical trap maybe used for manipulation of single molecule nucleic acid molecules forsequencing. (E.g., U.S. Pat. No. 5,776,674). Exemplary optical trappingsystems are commercially available from Cell Robotics, Inc.(Albuquerque, N. Mex.), S+L GmbH (Heidelberg, Germany) and P.A.L.M. Gmbh(Wolfratshausen, Germany).

Raman Labels

Certain embodiments of the invention may involve attaching a label tothe nucleotides to facilitate their measurement by the detection unit.Non-limiting examples of labels that could be used for Ramanspectroscopy include TRIT (tetramethyl rhodamine isothiol), NBD(7-nitrobenz-2-oxa-1,3-diazole), Texas Red dye, phthalic acid,terephthalic acid, isophthalic acid, cresyl fast violet, cresyl blueviolet, brilliant cresyl blue, para-aminobenzoic acid, erythrosine,biotin, digoxigenin, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,T-tetrachlorofluorescein,5-carboxyfluorescein, 5-carboxy rhodamine, 6-carboxyrhodamine,6-carboxytetramethyl amino phthalocyanines, azomethines, cyanines,xanthines, succinylfluoresceins and aminoacridine. These and other Ramanlabels may be obtained from commercial sources (e.g., Molecular Probes,Eugene, Oreg.).

Polycyclic aromatic compounds may function as Raman labels, as is knownin the art. Other labels that may be of use for particular embodimentsof the invention include cyanide, thiol, chlorine, bromine, methyl,phosphorus and sulfur. In certain embodiments of the invention, carbonnanotubes may be of use as Raman labels. The use of labels in Ramanspectroscopy is known (e.g., U.S. Pat. Nos. 5,306,403 and 6,174,677).The skilled artisan will realize that the Raman labels used shouldgenerate distinguishable Raman spectra and may be specifically bound toor associated with different types of nucleotides.

Labels may be attached directly to the nucleotides or may be attachedvia various linker compounds. Cross-linking reagents and linkercompounds of use in the disclosed methods are further described below.Alternatively, nucleotides that are covalently attached to Raman labelsare available from standard commercial sources (e.g., Roche MolecularBiochemicals, Indianapolis, Ind.; Promega Corp., Madison, Wis.; Ambion,Inc., Austin, Tex.; Amersham Pharmacia Biotech, Piscataway, N.J.). Ramanlabels that contain reactive groups designed to covalently react withother molecules, such as nucleotides, are commercially available (e.g.,Molecular Probes, Eugene, Oreg.). Methods for preparing labelednucleotides and incorporating them into nucleic acids are known (e.g.,U.S. Pat. Nos. 4,962,037; 5,405,747; 6,136,543; 6,210,896).

Nanoparticles

Certain embodiments of the invention involve the use of nanoparticles toenhance the Raman signal obtained from nucleotides. In some embodimentsof the invention, the nanoparticles are silver or gold nanoparticles,although any nanoparticles capable of providing a surface enhanced Ramanspectroscopy (SERS) signal may be used. In alternative embodiments ofthe invention, the nanoparticles may be nanoprisms (Jin et al., Science294:1902-3, 2001.) In various embodiments of the invention,nanoparticles of between 1 nm and 2 micrometers (μm) in diameter may beused. In alternative embodiments of the invention, nanoparticles ofbetween 2 nm to 1 μm, 5 nm to 500 nm, 5 nm to 200 nm, 10 nm to 200 nm,20 nm to 100 nm, 30 nm to 80 nm, 40 nm to 70 nm or 50 to 60 nm diameterare contemplated. In certain embodiments of the invention, nanoparticleswith an average diameter of 10 to 50 nm, 50 to 100 nm or about 100 nmare contemplated. The nanoparticles may be approximately spherical,rod-like, edgy, faceted or pointy in shape, although nanoparticles ofany shape or of irregular shape may be used. Methods of preparingnanoparticles are known (e.g., U.S. Pat. Nos. 6,054,495; 6,127,120;6,149,868; Lee and Meisel, J. Phys. Chem. 86:3391-3395, 1982; Jin etal., 2001). Nanoparticles may also be obtained from commercial sources(e.g., Nanoprobes Inc., Yaphank, N.Y.; Polysciences, Inc., Warrington,Pa.).

In certain embodiments of the invention, the nanoparticles may be singlenanoparticles and/or random aggregates of nanoparticles (colloidalnanoparticles). In other embodiments of the invention, nanoparticles maybe cross-linked to produce particular aggregates of nanoparticles, suchas dimers, trimers, tetramers or other aggregates. Certain alternativeembodiments may use heterogeneous mixtures of aggregates of differentsize, while other alternative embodiments may use homogenous populationsof nanoparticles. In certain embodiments, aggregates containing aselected number of nanoparticles (dimers, trimers, etc.) may be enrichedor purified by known techniques, such as ultracentrifugation in sucrosesolutions. In various embodiments of the invention, nanoparticleaggregates of about 100, 200, 300, 400, 500, 600, 700, 800, 900 to 1000nm in size or larger are contemplated.

Methods of cross-linking nanoparticles are known (e.g., Feldheim,“Assembly of metal nanoparticle arrays using molecular bridges,” TheElectrochemical Society Interface, Fall, 2001, pp. 22-25). Goldnanoparticles may be cross-linked, for example, using bifunctionallinker compounds bearing terminal thiol or sulfhydryl groups. Uponreaction with gold nanoparticles, the linker forms nanoparticle dimersthat are separated by the length of the linker. In other embodiments ofthe invention, linkers with three, four or more thiol groups may be usedto simultaneously attach to multiple nanoparticles (Feldheim, 2001). Theuse of an excess of nanoparticles to linker compounds prevents formationof multiple cross-links and nanoparticle precipitation. Aggregates ofsilver nanoparticles may be formed by standard synthesis methods knownin the art.

In alternative embodiments of the invention, the nanoparticles may bemodified to contain various reactive groups before they are attached tolinker compounds. Modified nanoparticles are commercially available,such as Nanogold® nanoparticles from Nanoprobes, Inc. (Yaphank, N.Y.).Nanogold® nanoparticles may be obtained with either single or multiplemaleimide, amine or other groups attached per nanoparticle. TheNanogold® nanoparticles are also available in either positively ornegatively charged form. Such modified nanoparticles may be attached toa variety of known linker compounds to provide dimers, trimers or otheraggregates of nanoparticles.

The type of linker compound used is not limiting, so long as it resultsin the production of small aggregates of nanoparticles that will notprecipitate in solution. In some embodiments of the invention, thelinker group may comprise phenylacetylene polymers (Feldheim, 2001).Alternatively, linker groups may comprise polytetrafluoroethylene,polyvinyl pyrrolidone, polystyrene, polypropylene, polyacrylamide,polyethylene or other known polymers. The linker compounds of use arenot limited to polymers, but may also include other types of moleculessuch as silanes, alkanes, derivatized silanes or derivatized alkanes.

In various embodiments of the invention, the nanoparticles may becovalently attached to nucleotides. In alternative embodiments of theinvention, the nucleotides may be directly attached to thenanoparticles, or may be attached to linker compounds that arecovalently or non-covalently bonded to the nanoparticles. In suchembodiments of the invention, rather than cross-linking two or morenanoparticles together the linker compounds may be used to attach anucleotide to a nanoparticle or a nanoparticle aggregate. In particularembodiments of the invention, the nanoparticles may be coated withderivatized silanes. Such modified silanes may be covalently attached tonucleotides using standard methods. Various methods known forcross-linking nucleic acids to surfaces discussed below may also be usedto attach nucleotides to nanoparticles. It is contemplated that thelinker compounds used to attach nucleotides may be of almost any length,ranging from about 0.05, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 30,35, 40, 45, 50, 55, 60, 65, 60, 80, 90 to 100 nm or even greater length.Certain embodiments of the invention may use linkers of heterogeneouslength.

In other embodiments of the invention, nucleotides may be adsorbed onthe surface of the nanoparticles or may be in close proximity to thenanoparticles (between about 0.2 and 1.0 nm). The skilled artisan willrealize that it covalent attachment of the nucleotides to nanoparticlesis not required in order to generate an enhanced Raman signal by SERS,SERRS or CARS.

In some embodiments of the invention, the nucleotides may be attached tonanoparticles as they travel down a microfluidic channel to formnucleotide-nanoparticle complexes. In certain embodiments of theinvention, the length of time available for the cross-linking reactionto occur may be limited. Such embodiments may utilize highly reactivecross-linking groups with rapid reaction rates, such as epoxide groups,azido groups, arylazido groups, triazine groups or diazo groups. Incertain embodiments of the invention, the cross-linking groups may bephotoactivated by exposure to intense light, such as a laser. Forexample, photoactivation of diazo or azido compounds results in theformation, respectively, of highly reactive carbene and nitrenemoieties. In certain embodiments, the reactive groups may be selected sothat they can only attach the nanoparticles to nucleotides, rather thancross-linking the nanoparticles to each other. The selection andpreparation of reactive cross-linking groups capable of binding tonucleotides is known in the art. In alternative embodiments of theinvention, nucleotides may themselves be covalently modified, forexample with a sulfhydryl group that can attach to gold nanoparticles.

In certain embodiments of the invention, nanoparticles may bemanipulated into microfluidic channels by any method known in the art,such as microfluidics, nanofluidics, hydrodynamic focusing orelectro-osmosis. In some embodiments, use of charged linker compounds orcharged nanoparticles may facilitate manipulation of nanoparticlesthrough the use of electrical gradients.

Immobilization of Nucleic Acids

In certain embodiments of the invention, one or more nucleic acidmolecules may be attached to a surface such as functionalized glass,silicon, silicate, PDMS (polydimethyl siloxane), polyvinylidenedifluoride (PVDF), silver or other metal coated surfaces, quartz,plastic, PTFE (polytetrafluoroethylene), PVP (polyvinyl pyrrolidone),poly(vinyl chloride), poly(methyl methacrylate), poly(dimethylsiloxane), polystyrene, polypropylene, polyacrylamide, latex, nylon,nitrocellulose, glass beads, magnetic beads, photopolymers which containphotoreactive species such as nitrenes, carbenes and ketyl radicalscapable of forming covalent links with nucleic acid molecules (see U.S.Pat. Nos. 5,405,766 and 5,986,076) or any other material known in theart that is capable of having functional groups such as amino, carboxyl,thiol, hydroxyl or Diels-Alder reactants incorporated on its surface.

In some embodiments of the invention, the surface functional groups maybe covalently attached to cross-linking compounds so that bindinginteractions between nucleic acid molecule and exonuclease and/orpolymerase may occur without steric hindrance. Typical cross-linkinggroups include ethylene glycol oligomers and diamines. Attachment may beby either covalent or non-covalent binding. Various methods of attachingnucleic acid molecules to surfaces are known in the art and may beemployed. In certain embodiments of the invention, the nucleic acidmolecule is fixed in place and immersed in a microfluidic flow down aflow path and/or microfluidic channel that transports the releasednucleotides past a detection unit. In non-limiting examples, themicrofluidic flow may result from a bulk flow of solvent down a flowpath and/or microfluidic channel.

In alternative embodiments of the invention, the bulk medium moves onlyslowly or not at all, but charged species within the solution (such asnegatively charged nucleotides) move down a flow path and/ormicrofluidic channel in response to an externally applied electricalfield.

Immobilization of nucleic acid molecules may be achieved by a variety ofknown methods. In an exemplary embodiment of the invention,immobilization may be achieved by coating a surface with streptavidin oravidin and the subsequent attachment of a biotinylated nucleic acid(Holmstrom et al., Anal. Biochem. 209:278-283, 1993). Immobilization mayalso occur by coating a silicon, glass or other surface with poly-L-Lys(lysine) or poly L-Lys, Phe (phenylalanine), followed by covalentattachment of either amino- or sulfhydryl-modified nucleic acids usingbifunctional crosslinking reagents (Running et al., BioTechniques8:276-277, 1990; Newton et al., Nucleic Acids Res. 21:1155-62, 1993).Amine residues may be coated on a surface through the use ofaminosilane.

Immobilization may take place by direct covalent attachment of5′-phosphorylated nucleic acids to chemically modified surfaces(Rasmussen et al., Anal. Biochem. 198:138-142, 1991). The covalent bondbetween the nucleic acid and the surface may be formed by condensationwith a water-soluble carbodiimide. This method facilitates apredominantly 5-attachment of the nucleic acids via their 5′-phosphates.

DNA is commonly bound to glass by first silanizing the glass surface,then activating with carbodiimide or glutaraldehyde. Alternativeprocedures may use reagents such as 3-glycidoxypropyltrimethoxysilane(GOP) or aminopropyltrimethoxysilane (APTS) with DNA linked via aminolinkers incorporated at either the 3′ or 5′ end of the molecule. DNA maybe bound directly to membrane surfaces using ultraviolet radiation.Other non-limiting examples of immobilization techniques for nucleicacids are disclosed in U.S. Pat. Nos. 5,610,287, 5,776,674 and6,225,068.

Bifunctional cross-linking reagents may be of use in various embodimentsof the invention, such as attaching a nucleic acid molecule to asurface. The bifunctional cross-linking reagents can be dividedaccording to the specificity of their functional groups, e.g., amino,guanidino, indole, or carboxyl specific groups. Exemplary methods forcross-linking molecules are disclosed in U.S. Pat. Nos. 5,603,872 and5,401,511. Cross-linking reagents include glutaraldehyde (GAD),bifunctional oxirane (OXR), ethylene glycol diglycidyl ether (EGDE), andcarbodiimides, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC).

Nucleic Acid Synthesis

Polymerases

Certain embodiments of the invention involve binding of a syntheticreagent, such as a DNA polymerase, to a primer molecule and the additionof Raman labeled nucleotides to the 3′ end of the primer. Non-limitingexamples of polymerases include DNA polymerases, RNA polymerases,reverse transcriptases, and RNA-dependent RNA polymerases. Thedifferences between these polymerases in terms of their “proofreading”activity and requirement or lack of requirement for primers and promotersequences are known in the art. Where RNA polymerases are used as thepolymerase, a template molecule to be sequenced may be double-strandedDNA. Non-limiting examples of polymerases include Thermatoga maritimaDNA polymerase, AmplitaqFS™ DNA polymerase, Taquenase™ DNA polymerase,ThermoSequenase™, Taq DNA polymerase, Qbeta™ replicase, T4 DNApolymerase, Thermus thermophilus DNA polymerase, RNA-dependent RNApolymerase and SP6 RNA polymerase.

A number of polymerases are commercially available, including Pwo DNAPolymerase (Boehringer Mannheim Biochemicals, Indianapolis, Ind.); BstPolymerase (Bio-Rad Laboratories, Hercules, Calif.); IsoTherm™ DNAPolymerase (Epicentre Technologies, Madison, Wis.); Moloney MurineLeukemia Virus Reverse Transcriptase, Pfu DNA Polymerase, AvianMyeloblastosis Virus Reverse Transcriptase, Thermus flavus (Tfl) DNAPolymerase and Thermococcus litoralis (Tli) DNA Polymerase (PromegaCorp., Madison, Wis.); RAV2 Reverse Transcriptase, HIV-1 ReverseTranscriptase, T7 RNA Polymerase, T3 RNA Polymerase, SP6 RNA Polymerase,E. coli RNA Polymerase, Thermus aquaticus DNA Polymerase, T7 DNAPolymerase +/−3′→5′ exonuclease, Klenow Fragment of DNA Polymerase I,Thermus ‘ubiquitous’ DNA Polymerase, and DNA polymerase I (AmershamPharmacia Biotech, Piscataway, N.J.). Any polymerase known in the artcapable of template dependent polymerization of labeled nucleotides maybe used. (See, e.g., Goodman and Tippin, Nat. Rev. Mol. Cell. Biol.1(2):101-9, 2000; U.S. Pat. No. 6,090,589.) Methods of using polymerasesto synthesize nucleic acids from labeled nucleotides are known (e.g.,U.S. Pat. Nos. 4,962,037; 5,405,747; 6,136,543; 6,210,896).

Primers

Generally, primers are between ten and twenty bases in length, althoughlonger primers may be employed. In certain embodiments of the invention,primers are designed to be complementary in sequence to a known portionof a template nucleic acid molecule. Known primer sequences may be used,for example, where primers are selected for identifying sequencevariants adjacent to known constant chromosomal sequences, where anunknown nucleic acid sequence is inserted into a vector of knownsequence, or where a native nucleic acid has been partially sequenced.Methods for synthesis of primers of any sequence are known. Otherembodiments of the invention involve sequencing a nucleic acid in theabsence of a known primer-binding site. In such cases, it may bepossible to use random primers, such as random hexamers or randomoligomers to initiate polymerization.

Nucleic Acid Digestion

In certain embodiments of the invention, methods of nucleic acidsequencing involve binding of an exonuclease or equivalent reagent tothe free end of a nucleic acid molecule and removal of nucleotides oneat a time. Non-limiting examples of nucleic acid digesting enzymes ofpotential use include E. coli exonuclease I, III, V or VII Bal 31exonuclease, mung bean nuclease, S1 nuclease, E. coli DNA polymerase Iholoenzyme or Klenow fragment, RecJ, exonuclease T, T4 or T7 DNApolymerase, Taq polymerase, exonuclease T7 gene 6, snake venomphosphodiesterase, spleen phosphodiesterase, Thermococcus litoralis DNApolymerase, Pyrococcus sp. GB-D DNA polymerase, lambda exonuclease, S.aureus micrococcal nuclease, DNase I, ribonuclease A, T1 micrococcalnuclease, or other exonucleases known in the art. Exonucleases areavailable from commercial sources such as New England Biolabs (Beverly,Mass.), Amersham Pharmacia Biotech (Piscataway, N.J.), Promega (Madison,Wis.), Sigma Chemicals (St. Louis, Mo.) or Boehringer Mannheim(Indianapolis, Ind.).

The skilled artisan will realize that enzymes with exonuclease activitymay remove nucleotides from the 5′ end, the 3′ end, or either end ofnucleic acid molecules. They can show specificity for RNA, DNA or bothRNA and DNA. Their activity may depend on the use of either single ordouble-stranded nucleic acids. They may be differentially affected bysalt concentration, temperature, pH, or divalent cations. These andother properties of exonucleases are known in the art. In certainembodiments of the invention, the rate of exonuclease activity may bemanipulated to coincide with the optimal rate of analysis of nucleotidesby the detection unit. Various methods are known for adjusting the rateof exonuclease activity, including adjusting the temperature, pressure,pH, salt or divalent cation concentration in a reaction chamber.

Although nucleoside monophosphates will generally be released fromnucleic acids by exonuclease activity, the embodiments of the inventionare not limited to detection of any particular form of free nucleotideor nucleoside but encompass any monomer that may be released from anucleic acid.

Reaction Chamber and Integrated Chip

Some embodiments of the invention concern apparatus comprising areaction chamber designed to contain an immobilization surface, nucleicacid molecule, exonuclease and nucleotides in an aqueous environment. Insome embodiments of the invention, the reaction chamber may betemperature controlled, for example by incorporation of Pelletierelements or other methods known in the art. Methods of controllingtemperature for low volume liquids are known. (See, e.g., U.S. Pat. Nos.5,038,853, 5,919,622, 6,054,263 and 6,180,372.)

In certain embodiments of the invention, the reaction chamber and anyassociated fluid channels, for example, a flow path, microfluidicchannels or channels to provide connections to waste ports, to a nucleicacid loading port, to a nanoparticle reservoir, to a source ofexonuclease or other fluid compartments are manufactured in a batchfabrication process, as known in the fields of computer chip manufactureand/or microcapillary chip manufacture. In some embodiments of theinvention, the reaction chamber and other components of the apparatus,such as the flow path and/or microfluidic channels, may be manufacturedas a single integrated chip. Such a chip may be manufactured by methodsknown in the art, such as by photolithography and etching. However, themanufacturing method is not limiting and other methods known in the artmay be used, such as laser ablation, injection molding, casting,molecular beam epitaxy, dip-pen nanolithograpy, chemical vapordeposition (CVD) fabrication, electron beam or focused ion beamtechnology or imprinting techniques. Methods for manufacture ofnanoelectromechanical systems may be used for certain embodiments of theinvention. (See, e.g., Craighead, Science 290:1532-36, 2000.)Microfabricated chips are commercially available from, e.g., CaliperTechnologies Inc. (Mountain View, Calif.) and ACLARA BioSciences Inc.(Mountain View, Calif.).

To facilitate detection of nucleotides by the detection unit thematerial comprising the flow path or flow-through cell may be selectedto be transparent to electromagnetic radiation at the excitation andemission frequencies used for the detection unit. Glass, silicon, andany other materials that are generally transparent in the wavelengthsused for Raman spectroscopy may be used. In some embodiments of theinvention the surfaces of the flow path or flow-through cell that areopposite the detection unit may be coated with silver, gold, platinum,copper, aluminum or other materials that are relatively opaque to thedetection unit. In that position, the opaque material is available toenhance the Raman signal, for example by SERS, while not interferingwith the function of the detection unit. Alternatively, the flow path orflow-through cell may contain a mesh comprising silver, gold, platinum,copper, aluminum or other Raman signal enhancing metal. In otheralternative embodiments of the invention, the flow path or flow-throughcell may contain metal nanoparticles.

Flow Path and Microfluidic Channels

In certain embodiments of the invention, the nucleotides released from anucleic acid are moved down a flow path and/or microfluidic channelspast a detection unit. A non-limiting example of techniques fortransport of nucleotides includes microfluidic techniques. The flow pathand/or microfluidic channels can comprise a microcapillary (e.g., fromACLARA BioSciences Inc., Mountain View, Calif.) or a liquid integratedcircuit (e.g., Caliper Technologies Inc., Mountain View, Calif.). Amicrochannel flow path may be from about 5 to 200 μm in diameter, with adiameter of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 μm.

In certain embodiments of the invention, the nucleotides to be detectedmove down the flow path and/or microfluidic channels by bulk flow ofsolvent. In other embodiments of the invention, microcapillaryelectrophoresis may be used to transport nucleotides down the flow pathand/or microfluidic channels. Microcapillary electrophoresis generallyinvolves the use of a thin capillary or channel that may or may not befilled with a particular separation medium. Electrophoresis ofappropriately charged molecular species, such as negatively chargednucleotides, occurs in response to an imposed electrical field, negativeon the reaction chamber side of the apparatus and positive on thedetection unit side. Although electrophoresis is often used for sizeseparation of a mixture of components that are simultaneously added tothe microcapillary, it can also be used to transport similarly sizednucleotides that are sequentially released from a nucleic acid. Becausethe purine nucleotides (A, G) are larger than the pyrimidine nucleotides(C, T, U) and would therefore migrate more slowly, the length of theflow path and/or microfluidic channels and the corresponding transittime past the detection unit may kept to a minimum to preventdifferential migration from mixing up the order of nucleotides releasedfrom the nucleic acid. Alternatively, the medium filling themicrocapillary may be selected so that the migration rates of purine andpyrimidine nucleotides down the flow path and/or microfluidic channelsare similar or identical. Methods of microcapillary electrophoresis havebeen disclosed, for example, by Woolley and Mathies (Proc. Natl. Acad.Sci. USA 91:11348-352, 1994).

In certain embodiments of the invention, flow paths and/or microfluidicchannels may contain aqueous solutions with relatively high viscosity,such as glycerol solutions. Such high viscosity solutions may serve todecrease the flow rate and increase the reaction time available, forexample, for cross-linking nucleotides to nanoparticles.

Microfabrication of microfluidic devices, including microcapillaryelectrophoretic devices has been disclosed in, e.g., Jacobsen et al.(Anal. Biochem, 209:278-283, 1994); Effenhauser et al. (Anal. Chem.66:2949-2953, 1994); Harrison et al. (Science 261:895-897, 1993) andU.S. Pat. No. 5,904,824. These methods may comprise micromoldingtechniques with silicon masters made using standard photolithography orfocused ion beam techniques, or photolithographic etching of micronscale channels on silica, silicon or other crystalline substrates orchips. Such techniques may be readily adapted for use in the disclosedmethods and apparatus. In some embodiments of the invention, themicrocapillary may be fabricated from the same materials used forfabrication of a reaction chamber, using techniques known in the art.

Detection Unit

In various embodiments of the invention, the detection unit is designedto detect and quantify nucleotides by Raman spectroscopy. Methods fordetection of micromolar concentrations of nucleotides by Ramanspectroscopy are known in the art. (See, e.g., U.S. Pat. Nos. 5,306,403;6,002,471; 6,174,677). Exemplary methods for detection of nucleotides atthe single molecule level are disclosed in the Examples below.Variations on surface enhanced Raman spectroscopy (SERS), surfaceenhanced resonance Raman spectroscopy (SERRS) and coherent anti-StokesRaman spectroscopy (CARS) have been disclosed. The sensitivity of Ramandetection is enhanced by a factor of 10⁶ or more for molecules adjacentto roughened metal surfaces, such as silver, gold, platinum, copper oraluminum surfaces.

A non-limiting example of a Raman detection unit is disclosed in U.S.Pat. No. 6,002,471. An excitation beam is generated by either afrequency doubled Nd:YAG laser at 532 nm wavelength or a frequencydoubled Ti:sapphire laser at 365 nm wavelength. Pulsed laser beams orcontinuous laser beams may be used. The excitation beam passes throughconfocal optics and a microscope objective, and is focused onto the flowpath and/or the flow-through cell. The Raman emission light from thenucleotides is collected by the microscope objective and the confocaloptics and is coupled to a monochromator for spectral dissociation. Theconfocal optics includes a combination of dichroic filters, barrierfilters, confocal pinholes, lenses, and mirrors for reducing thebackground signal. Standard full field optics can be used as well asconfocal optics. The Raman emission signal is detected by a Ramandetector, comprising an avalanche photodiode interfaced with a computerfor counting and digitization of the signal.

Another example of a Raman detection unit is disclosed in U.S. Pat. No.5,306,403, including a Spex Model 1403 double-grating spectrophotometerwith a gallium-arsenide photomultiplier tube (RCA Model C31034 or BurleIndustries Model C3103402) operated in the single-photon counting mode.The excitation source comprises a 514.5 nm line argon-ion laser fromSpectraPhysics, Model 166, and a 647.1 nm line of a krypton-ion laser(Innova 70, Coherent).

Alternative excitation sources include a nitrogen laser (Laser ScienceInc.) at 337 nm and a helium-cadmium laser (Liconox) at 325 nm (U.S.Pat. No. 6,174,677), a light emitting diode, an Nd:YLF laser, and/orvarious ions lasers and/or dye lasers. The excitation beam may bespectrally purified with a bandpass filter (Corion) and may be focusedon the flow path and/or flow-through cell using a 6× objective lens(Newport, Model L6X). The objective lens may be used to both excite thenucleotides and to collect the Raman signal, by using a holographic beamsplitter (Kaiser Optical Systems, Inc., Model HB 647-26N18) to produce aright-angle geometry for the excitation beam and the emitted Ramansignal. A holographic notch filter (Kaiser Optical Systems, Inc.) may beused to reduce Rayleigh scattered radiation. Alternative Raman detectorsinclude an ISA HR-320 spectrograph equipped with a red-enhancedintensified charge-coupled device (RE-ICCD) detection system (PrincetonInstruments). Other types of detectors may be used, such asFourier-transform spectrographs (based on Michaelson interferometers),charged injection devices, photodiode arrays, InGaAs detectors,electron-multiplied CCD, intensified CCD and/or phototransistor arrays.

Any suitable form or configuration of Raman spectroscopy or relatedtechniques known in the art may be used for detection of nucleotides,including but not limited to normal Raman scattering, resonance Ramanscattering, surface enhanced Raman scattering, surface enhancedresonance Raman scattering, coherent anti-Stokes Raman spectroscopy(CARS), stimulated Raman scattering, inverse Raman spectroscopy,stimulated gain Raman spectroscopy, hyper-Raman scattering, molecularoptical laser examiner (MOLE) or Raman microprobe or Raman microscopy orconfocal Raman microspectrometry, three-dimensional or scanning Raman,Raman saturation spectroscopy, time resolved resonance Raman, Ramandecoupling spectroscopy or UV-Raman microscopy.

Information Processing and Control System and Data Analysis

In certain embodiments of the invention, the nucleic acid sequencingapparatus may comprise an information processing system. The disclosedmethods and apparatus are not limiting for the type of informationprocessing system used. An exemplary information processing system mayincorporate a computer comprising a bus for communicating informationand a processor for processing information. In one embodiment of theinvention, the processor is selected from the Pentium® family ofprocessors, including without limitation the Pentium® II family, thePentium® DI family and the Pentium® 4 family of processors availablefrom Intel Corp. (Santa Clara, Calif.). In alternative embodiments ofthe invention, the processor may be a Celeron®, an Itanium®, or aPentium Xeon® processor (Intel Corp., Santa Clara, Calif.). In variousother embodiments of the invention, the processor may be based on Intel®architecture, such as Intel® IA-32 or Intel® IA-64 architecture.Alternatively, other processors may be used. The information processingand control system may further comprise any peripheral devices known inthe art, such as memory, display, keyboard and/or other devices.

In particular embodiments of the invention, the detection unit may beoperably coupled to the information processing system. Data from thedetection unit may be processed by the processor and data stored inmemory. Data on emission profiles for standard nucleotides may also bestored in memory. The processor may compare the emission spectra fromnucleotides in the flow path and/or flow-through cell to identify thetype of nucleotide released from the nucleic acid molecule. The memorymay also store the sequence of nucleotides released from the nucleicacid molecule. The processor may analyze the data from the detectionunit to determine the sequence of the nucleic acid. The informationprocessing system may also perform standard procedures such assubtraction of background signals and “base-calling” determination whenoverlapping signals are detected.

While the disclosed methods may be performed under the control of aprogrammed processor, in alternative embodiments of the invention, themethods may be fully or partially implemented by any programmable orhardcoded logic, such as Field Programmable Gate Arrays (FPGAs), TTLlogic, or Application Specific Integrated Circuits (ASICs).Additionally, the disclosed methods may be performed by any combinationof programmed general purpose computer components and/or custom hardwarecomponents.

Following the data gathering operation, the data will typically bereported to a data analysis operation. To facilitate the analysisoperation, the data obtained by the detection unit will typically beanalyzed using a digital computer such as that described above.Typically, the computer will be appropriately programmed for receipt andstorage of the data from the detection unit as well as for analysis andreporting of the data gathered.

In certain embodiments of the invention, custom designed softwarepackages may be used to analyze the data obtained from the detectionunit. In alternative embodiments of the invention, data analysis may beperformed, using an information processing system and publicly availablesoftware packages. Non-limiting examples of available software for DNAsequence analysis include the PRISM™ DNA Sequencing Analysis Software(Applied Biosystems, Foster City, Calif.), the Sequencher™ package (GeneCodes, Ann Arbor, Mich.), and a variety of software packages availablethrough the National Biotechnology Information Facility at websitewww.nbif.org/links/1.4.1.php.

EXAMPLES Example 1 Raman Detection of Nucleotides

Methods and Apparatus

In a non-limiting example, the excitation beam of a Raman detection unitwas generated by a titanium:sapphire laser (Mira by Coherent) at anear-infrared wavelength (750˜950 nm) or a gallium aluminum arsenidediode laser (PI-ECL series by Process Instruments) at 785 nm or 830 nm.Pulsed laser beams or continuous beams were used. The excitation beamwas reflected by a dichroic mirror (holographic notch filter by KaiserOptical or a dichromatic interference filter by Chroma or Omega Optical)into a collinear geometry with the collected beam. The reflected beampassed through a microscope objective (Nikon LU series), and was focusedonto the Raman active substrate where target analytes (nucleotides orpurine or pyrimidine bases) were located.

The Raman scattered light from the analytes was collected by the samemicroscope objective, and passed the dichroic mirror to the Ramandetector. The Raman detector comprised a focusing lens, a spectrograph,and an array detector. The focusing lens focused the Raman scatteredlight through the entrance slit of the spectrograph. The spectrograph(Acton Research) comprised a grating that dispersed the light by itswavelength. The dispersed light was imaged onto an array detector(back-illuminated deep-depletion CCD camera by RoperScientific). Thearray detector was connected to a controller circuit, which wasconnected to a computer for data transfer and control of the detectorfunction.

For surface-enhanced Raman spectroscopy (SERS), the Raman activesubstrate consisted of metallic nanoparticles or metal-coatednanostructures. Silver nanoparticles, ranging in size from 5 to 200 nm,was made by the method of Lee and Meisel (J. Phys. Chem., 86:3391,1982). Alternatively, samples were placed on an aluminum substrate underthe microscope objective. The Figures discussed below were collected ina stationary sample on the aluminum substrate. The number of moleculesdetected was determined by the optical collection volume of theilluminated sample.

The ability to detect single nucleotides by SERS was confirmed using a100 μm or 200 μm microfluidic channel. In various embodiments of theinvention, nucleotides may be delivered to a Raman active substratethrough a microfluidic channel (between about 5 and 200 μm wide).Microfluidic channels can be made by molding polydimethylsiloxane(PDMS), using the technique disclosed in Anderson et al. (“Fabricationof topologically complex three-dimensional microfluidic systems in PDMSby rapid prototyping,” Anal. Chem. 72:3158-3164, 2000).

Where SERS was performed in the presence of silver nanoparticles, thenucleotide, purine or pyrimidine analyte was mixed with LiCl (90 μMfinal concentration) and nanoparticles (0.25 M final concentrationsilver atoms). SERS data were collected using room temperature analytesolutions.

Results

Nucleoside monophosphates, purine bases and pyrimidine bases wereanalyzed by SERS, using the system disclosed above. Table 1 shows thepresent detection limits for various analytes of interest.

TABLE 1 SERS Detection of Nucleoside Monophosphates, Purines andPyrimidines Number of Molecules Analyte Final Concentration DetecteddAMP 9 picomolar (pM) ~1 molecule Adenine 9 pM ~1 molecule dGMP 90 μM 6× 10⁶ Guanine 909 pM 60 dCMP 909 μM 6 × 10⁷ Cyotosine 90 nM 6 × 10³ dTMP9 μM 6 × 10⁵ Thymine 90 nM 6 × 10³

Conditions were optimized for adenine nucleotides only. LiCL (90 μMfinal concentration) was determined to provide optimal SERS detection ofadenine nucleotides. Detection of other nucleotides may be facilitatedby use of other alkali-metal halide salts, such as NaCl, KCl, RbCl orCsCl. The claimed methods are not limited by the electrolyte solutionused, and it is contemplated that other types of electrolyte solutions,such as MgCl, CaCl, NaF, KBr, LiI, etc. may be of use. The skilledartisan will realize that electrolyte solutions that do not exhibitstrong Raman signals will provide minimal interference with SERSdetection of nucleotides. The results demonstrate that the Ramandetection system and methods disclosed above were capable of detectingand identifying single molecules of nucleotides and purine bases. Thisis the first report of Raman detection of unlabeled nucleotides at thesingle nucleotide level.

Example 2 Raman Emission Spectra of Nucleotides, Purines and Pyrimidines

The Raman emission spectra of various analytes of interest was obtainedusing the protocol of Example 1, with the indicated modifications. FIG.4 shows the Raman emission spectra of a 100 mM solution of each of thefour nucleoside monophosphates, in the absence of surface enhancementand without Raman labels. No LiCl was added to the solution. A 100millisecond (msec) data collection time was used. Lower concentrationsof nucleotides may be detected with longer collection times. Excitationoccurred at 514 nm. For each of the following figures, a 785 nmexcitation wavelength was used. As shown in FIG. 4, the unenhanced Ramanspectra showed characteristic emission peaks for each of the fourunlabeled nucleoside monophosphates.

FIG. 5 shows the SERS spectrum of a 1 nm solution of guanine, in thepresence of LiCl and silver nanoparticles. Guanine was obtained fromdGMP by acid treatment, as discussed in Nucleic Acid Chemistry, Part 1,L. B. Townsend and R. S. Tipson (eds.), Wiley-Interscience, New York,1978. The SERS spectrum was obtained using a 100 msec data collectiontime.

FIG. 6 shows the SERS spectrum of a 10 nM cytosine solution, obtainedfrom dCMP by acid hydrolysis. Data were collected using a 1 secondcollection time.

FIG. 7 shows the SERS spectrum of a 100 nM thymine solution, obtained byacid hydrolysis of dTMP. Data were collected using a 100 msec collectiontime.

FIG. 8 shows the SERS spectrum of a 100 pM adenine solution, obtained byacid hydrolysis of dAMP. Data were collected for 1 second.

FIG. 9 shows the SERS spectrum of a 500 nM solution of dATP (lowertrace) and fluorescein-labeled dATP (upper trace). dATP-fluorescein waspurchased from Roche Applied Science (Indianapolis, Ind.). The Figureshows a strong increase in SERS signal due to labeling with fluorescein.

Example 3 SERS Detection of Nucleotides and Amplification Products

Silver Nanoparticle Formation

Silver nanoparticles used for SERS detection were produced according toLee and Meisel (1982). Eighteen milligrams of AgNO₃ were dissolved in100 mL (milliliters) of distilled water and heated to boiling. Ten mL ofa 1% sodium citrate solution was added drop-wise to the AgNO₃ solutionover a 10 min period. The solution was kept boiling for another hour.The resulting silver colloid solution was cooled and stored.

SERS Detection of Adenine

The Raman detection system was as disclosed in Example 1. One mL ofsilver colloid solution was diluted with 2 mL of distilled water. Thediluted silver colloid solution (160 μL) (microliters) was mixed with 20μL of a 10 nM (nanomolar) adenine solution and 40 μL of LiCl (0.5 molar)on an aluminum tray. The LiCl acted as a Raman enhancing agent foradenine. The final concentration of adenine in the sample was 0.9 nM, ina detection volume of about 100 to 150 femtoliters, containing anestimated 60 molecules of adenine. The Raman emission spectrum wascollected using an excitation source at 785 nm excitation, with a 100millisecond collection time. As shown in FIG. 10, this proceduredemonstrated the detection of 60 molecules of adenine, with strongemission peaks detected at about 833 nm and 877 nm. As discussed inExample 1, single molecule detection of adenine has been shown using thedisclosed methods and apparatus.

Rolling Circle Amplification

One picomole (pmol) of a rolling circle amplification (RCA) primer wasadded to 0.1 pmol of circular, single-stranded M13 DNA template. Themixture was incubated with 1× T7 polymerase 160 buffer (20 mM(millimolar) Tris-HCl, pH 7.5, 10 mM MgCl₂, 1 mM dithiothreitol), 0.5 mMdNTPs and 2.5 units of T7 DNA polymerase for 2 hours at 37° C.,resulting in formation of an RCA product. A negative control wasprepared by mixing and incubating the same reagents without the DNApolymerase.

SERS Detection of RCA Product

One μL of the RCA product and 1 μL of the negative control sample wereseparately spotted on an aluminum tray and air-dried. Each spot wasrinsed with 5 μL of 1×PBS (phosphate buffered saline). The rinse wasrepeated three times and the aluminum tray was air-dried after the finalrinse.

One milliliter of silver colloid solution prepared as above was dilutedwith 2 mL of distilled water. Eight microliters of the diluted silvercolloid solution was mixed with 2 μL of 0.5 M LiCl and added to the RCAproduct spot on the aluminum tray. The same solution was added to thenegative control spot. The Raman signals were collected as disclosedabove. As demonstrated in FIG. 11, an RCA product was detectable bySERS, with emission peaks at about 833 and 877 nm. Under the conditionsof this protocol, with an LiCl enhancer, the signal strength from theadenine moieties is stronger than those for guanine, cytosine andthymine. The negative control (not shown) showed that the Raman signalwas specific for the RCA product, as no signal was observed in theabsence of amplification.

Example 4 Exonuclease Digestion of Nucleic Acids

Exonuclease treatment is performed according to Sauer et al. (J.Biotech. 86:181-201, 2001). Single nucleic acid molecules labeled on the5′ end with biotin are prepared by PCR amplification of a nucleic acidtemplate, using a 5′-biotinylated oligonucleotide primer. A cone-shaped3 μm single-mode optical fiber (SMC-A0630B, Laser Components GmbH,Olching, Germany) is prepared. The glass fiber is chemically etched withHF to form a sharp tip. After coating with3-mercaptopropyltrimethoxysilane, the tip is treated withγ-maleinimidobutyric acid N-hydroxysuccinamide (GMBS). The tip of thefiber is activated with streptavidin and allowed to bind to thebiotinylated DNA. Unbound DNA is removed by washing.

The fiber containing a single molecule of bound DNA is inserted into aPDMS reaction chamber attached to a 5 μm microchannel. Exonuclease I isadded to the reaction chamber to initiate cleavage of the ssDNA. Theexonuclease is confined to the reaction chamber by use of an opticaltrap (e.g. Walker et al., FEBS Lett. 459:39-42, 1999; Bennink et al.,Cytometry 36:200-208, 1999; Mehta et al., Science 283:1689-95, 1999;Smith et al., Am. J. Phys. 67:26-35, 1999). Optical trapping devices areavailable from Cell Robotics, Inc. (Albuquerque, N. Mex.), S+L GmbH(Heidelberg, Germany) and P.A.L.M. Gmbh (Wolfratshausen, Germany).Nucleoside monophosphates are released by exonuclease digestion andtransported past a Raman detector, as disclosed in Example 1, bymicrofluidic flow. A 90 μM concentration of LiCl is added to thedetection mixture, and the microfluidic channel in the vicinity of thedetector is packed with silver nanoparticles prepared according to Leeand Meisel (1982). Single nucleotides are detected as they flow past theRaman detector, allowing determination of the nucleic acid sequence.

Example 5 Nucleic Acid Sequencing Using Raman-Labeled Nucleotides

Certain embodiments of the invention are exemplified in FIG. 1. FIG. 1illustrates methods and an apparatus 10 for sequencing individualsingle-stranded nucleic acid molecules 13 that are attached to animmobilization surface 14 in a reaction chamber 11 and disassembled in adeconstruction reaction. In such embodiments of the invention, thereaction chamber 11 contains one or more exonucleases 15 thatsequentially remove one nucleotide 16 at a time from the unattached end17 of the nucleic acid molecule 13.

As the nucleotides 16 are released, they move down a flow path 12 past adetection unit 18. The detection unit 18 comprises an excitation source19, such as a laser, that emits an excitatory beam 20. The excitatorybeam 20 interacts with the released nucleotides 16 so that electrons areexcited to a higher energy state. The Raman emission spectrum thatresults from the return of the electrons to a lower energy state isdetected by a Raman spectroscopic detector 21, such as a spectrometer, amonochromator or a charge coupled device (CCD), such as a CCD camera.

Preparation of Reaction Chamber and Flow Path

Borofloat glass wafers (Precision Glass & Optics, Santa Ana, Calif.) arepre-etched for a short period in concentrated HF (hydrofluoric acid) andcleaned before deposition of an amorphous silicon sacrificial layer in aplasma-enhanced chemical vapor deposition (PECVD) system (PER-A,Technics West, San Jose, Calif.). Wafers are primed withhexamethyldisilazane (HMDS), spin-coated with photoresist (Shipley 1818,Marlborough, Mass.) and soft-baked. A contact mask aligner (QuintelCorp. San Jose, Calif.) is used to expose the photoresist layer with oneor more mask designs, and the exposed photoresist removed using amixture of Microposit developer concentrate (Shipley) and water.Developed wafers are hard-baked and the exposed amorphous siliconremoved using CF₄ (carbon tetrafluoride) plasma in a PECVD reactor.Wafers are chemically etched with concentrated HF to produce thereaction chamber 11 and flow path 12. The remaining photoresist isstripped and the amorphous silicon removed. Using these methods,microchannels of about 50 to 100 μm diameter may be prepared. Smallerdiameter channels may be prepared by known methods, such as coating theinside of the microchannel to narrow the diameter, or usingnanolithography, focused electron beam, focused ion beam or focused atomlaser techniques. Methods for making PDMS microchannels are discussedabove.

Access holes are drilled into the etched wafers with a diamond drill bit(Crystalite, Westerville, Ohio). A finished chip is prepared bythermally bonding two complementary etched and drilled plates to eachother in a programmable vacuum furnace (Centurion VPM, J. M. Ney,Yucaipa, Calif.). Alternative exemplary methods for fabrication of achip incorporating a reaction chamber 11 and flow path 12 are disclosedin U.S. Pat. Nos. 5,867,266 and 6,214,246. In certain embodiments of theinvention, a nylon filter with a molecular weight cutoff of 2,500daltons is inserted between the reaction chamber 11 and the flow path 12to prevent exonuclease 15 from leaving the reaction chamber 11.

Nucleic Acid Preparation and Exonuclease Treatment

Human chromosomal DNA is purified according to Sambrook et al. (1989).Following digestion with Bam H1, the genomic DNA fragments are insertedinto the multiple cloning site of the pBluescript® II phagemid vector(Stratagene, Inc., La Jolla, Calif.) and grown up in E. coli. Afterplating on ampicillin-containing agarose plates a single colony isselected and grown up for sequencing. Single-stranded DNA copies of thegenomic DNA insert are rescued by co-infection with helper phage. Afterdigestion in a solution of proteinase K:sodium dodecyl sulphate (SDS),the DNA is phenol extracted and then precipitated by addition of sodiumacetate (pH 6.5, about 0.3 M) and 0.8 volumes of 2-propanol. The DNAcontaining pellet is resuspended in Tris-EDTA buffer and stored at −20°C. until use. Agarose gel electrophoresis shows a single band ofpurified DNA.

M13 forward primers complementary to the known pBluescript® sequence,located next to the genomic DNA insert, are purchased from MidlandCertified Reagent Company (Midland, Tex.). The primers are covalentlymodified to contain a biotin moiety attached to the 5′ end of theoligonucleotide. The biotin group is covalently linked to the5′-phosphate of the primer via a (CH₂)₆ spacer. Biotin-labeled primersare allowed to hybridize to the ssDNA template molecules prepared fromthe pBluescript® vector. The primer-template complexes are then attachedto streptavidin-coated beads 14 according to Dorre et al. (Bioimaging 5:139-152, 1997). At appropriate DNA dilutions, a single primer-templatecomplex is attached to a single bead 14. A bead 14 containing a singleprimer-template complex is inserted into the reaction chamber 11 of asequencing apparatus 10.

The primer-template is incubated with modified T7 DNA polymerase (UnitedStates Biochemical Corp., Cleveland, Ohio). The reaction mixturecontains unlabeled deoxyadenosine-5′-triphosphate (dATP) anddeoxyguanosine-5′-triphosphate (dGTP), digoxigenin-labeleddeoxyuridine-5′-triphosphate (digoxigenin-dUTP) and rhodamine-labeleddeoxycytidine-5′-triphosphate (rhodamine-dCTP). The polymerizationreaction is allowed to proceed for 2 hours at 37° C. After synthesis ofthe digoxigenin and rhodamine labeled nucleic acid 13, the templatestrand is separated from the labeled nucleic acid 13, and the templatestrand, DNA polymerase and unincorporated nucleotides are washed out ofthe reaction chamber 11.

Exonuclease 15 activity is initiated by addition of exonuclease III 15to the reaction chamber 11. The reaction mixture is maintained at pH 8.0and 37° C. As nucleotides 16 are released from the 3′ end 17 of thenucleic acid 13, they are transported by microfluidic flow down the flowpath 12 past the detection unit 18.

Example 6 Nucleic Acid Sequencing Using Covalent Attachment toNanoparticles

Another exemplary embodiment of the invention is disclosed in FIG. 2.Nucleotides 130 are released from a nucleic acid by exonuclease activityas discussed above. In certain embodiments of the invention, thenucleotides 130 are unlabeled. Unlabeled nucleic acids directly purifiedfrom any organ, tissue and/or cell sample or obtained by known cloningmethods may be sequenced using exonuclease treatment. Releasednucleotides 130 travel down a microfluidic channel 110.

The released nucleotides 130 are mixed with silver nanoparticles 140,prepared according to Lee and Meisel (J. Phys. Chem. 86:3391-3395,1982). The nanoparticles are 5 to 200 nm in size. Prior to exposure tonucleotides 130, surface-modified nanoparticles 140 are coated with asilane, such as 3-glycidoxypropyltrimethoxysilane (GOP), a reactivelinker compound. GOP contains a terminal highly reactive epoxide group.The silanized nanoparticles 140 are mixed with nucleotides 130 andallowed to form covalent cross-links with the nucleotides 130. Thenucleotide-nanoparticle complexes 150 pass through a flow through cell170 and are identified by SERS, SERRS and/or CARS using a Ramandetection unit 180. Because of the close proximity of the nucleotides130 to the nanoparticles 140, the Raman signals are greatly enhanced,allowing detection of single nucleotides 130 passing through theflow-through cell 170.

Example 7 Apparatus for Nucleic Acid Sequencing

FIG. 3 shows another exemplary embodiment of the invention. A DNAsequencing apparatus 210 comprises a reaction chamber 220 in fluidcommunication with an influx channel 230 and an efflux channel 240.Fluid movement may be controlled through the use of one or more valves250. A microfluidic channel 260 is also in fluid communication with thereaction chamber 220. Nucleotides released from one or more nucleicacids by exonuclease activity exit the reaction chamber 220 through themicrofluidic channel 260. The nucleotides are mixed with nanoparticlesthat move through a nanoparticle channel 270 in fluid communication withthe microfluidic channel 260. Covalent attachment of nucleotides tonanoparticles occurs within an attachment channel 280. The covalentlybound nucleotide-nanoparticle complexes pass through a flow-through cell290 where the nucleotides are identified by a Raman detection unit 300.The detection unit 300 comprises a laser 320 and Raman detector 310. Thelaser emits an excitation beam 330 that excites nucleotides within theflow-through cell 290. Excited nucleotides emit a Raman signal that isdetected by the Raman detector 310.

In certain embodiments of the invention, nanoparticles may be recoveredin a recycling chamber 340. The nanoparticles are chemically treated,for example with acid solutions, and then washed to remove boundnucleotides, linker compounds and any other attached or adsorbedmolecules. The nanoparticles may be recycled to a nanoparticle reservoir370 via a recycling channel 360. In some embodiments of the invention,nanoparticles may be coated with a linker compound, such as GOP, in therecycling channel 360 and/or the nanoparticle reservoir 370. Wasteeffluent is removed from the recycling chamber 340 via a waste channel350.

All of the METHODS and APPARATUS disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. It will be apparent to those of skill in the art thatvariations may be applied to the METHODS and APPARATUS described hereinwithout departing from the concept, spirit and scope of the claimedsubject matter. More specifically, it will be apparent that certainagents that are both chemically and physiologically related may besubstituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the claimed subject matter.

1. A method comprising: a) using an exonuclease to release unlabelednucleotides from one end of one or more nucleic acid molecules; b)separating the unlabeled nucleotides from the exonuclease and the one ormore nucleic acid molecules; c) identifying the unlabeled nucleotides byRaman spectroscopy to determine identified nucleotides; and d)determining the sequence of the nucleic acid from the identifiednucleotides.
 2. The method of claim 1, wherein single molecules of theunlabeled nucleotides are identified by Raman spectroscopy.
 3. Themethod of claim 2, wherein a single nucleic acid molecule is sequenced.4. The method of claim 1, wherein multiple nucleic acid molecules aresequenced simultaneously.
 5. The method of claim 1, wherein the one ormore nucleic acid molecules is attached to a surface.
 6. The method ofclaim 1, wherein the unlabeled nucleotides are identified by surfaceenhanced Raman spectroscopy (SERS), surface enhanced resonance Ramanspectroscopy (SERRS) and/or coherent anti-Stokes Raman spectroscopy(CARS). 7-13. (canceled)
 14. The method of claim 1, wherein theunlabeled nucleotides are attached to nanoparticles after thenucleotides are removed from the nucleic acid.
 15. The method of claim1, wherein the unlabeled nucleotides are attached to nanoparticlesbefore the nucleotides are removed from the nucleic acid.
 16. The methodof claim 15, wherein nanoparticles are attached to the 3′ end of thenucleic acid. 17-21. (canceled)
 22. A method comprising: a) obtainingnucleotides that are attached to Raman labels; b) synthesizing a nucleicacid comprising labeled nucleotides; c) removing nucleotides from oneend of the nucleic acid; d) identifying the nucleotides by Ramanspectroscopy; and e) determining the sequence of the nucleic acid. 23.The method of claim 22, wherein single nucleotide molecules areidentified by Raman spectroscopy
 24. The method of claim 22, whereineach type of nucleotide is labeled with a distinguishable Raman label.25. The method of claim 22, wherein only pyrimidine nucleotides arelabeled with Raman labels.
 26. The method of claim 22, furthercomprising: (i) obtaining at least one template nucleic acid molecule;(ii) hybridizing the template nucleic acid molecule to a primer; and(iii) adding a DNA polymerase to synthesize said nucleic acid.
 27. Anapparatus comprising: a) a reaction chamber; b) a microfluidic channelin fluid communication with the reaction chamber; c) a flow-through cellin fluid communication with the microfluidic channel; and d) a Ramandetection unit operably coupled to the flow-through cell.
 28. Theapparatus of claim 27, wherein the Raman detector is capable ofdetecting single molecules of nucleotides.
 29. The apparatus of claim28, wherein the nucleotides are unlabeled.
 30. The apparatus of claim27, further comprising nanoparticles in the flow-through cell.